CN114805316B

CN114805316B - Diketoazepine compound or pharmaceutically acceptable salts and tautomers thereof, preparation method, pharmaceutical composition and application thereof

Info

Publication number: CN114805316B
Application number: CN202210606356.4A
Authority: CN
Inventors: 蒋晟; 肖易培; 张阔军; 郝海平; 唐鹤; 王天雨; 李茂天; 倪勇; 章翔宇; 王淋
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2022-05-31
Filing date: 2022-05-31
Publication date: 2024-04-26
Anticipated expiration: 2042-05-31
Also published as: CN114805316A

Abstract

The invention discloses a diketone nitrogen heterocyclic compound with a structure shown in a general formula I or pharmaceutically acceptable salts, tautomers, metabolites, prodrugs, solvates or hydrates thereof, a pharmaceutical composition and application thereof; the invention overcomes the defects of single structure, lack of non-covalent and non-peptide high-efficiency small molecule inhibitors and the like of the prior broad-spectrum antiviral drugs, provides the compound shown in the formula I, has good inhibitory activity on 3C-like cysteine protease, has small toxic and side effects and has good therapeutic effect on infectious diseases.

Description

Diketoazepine compound or pharmaceutically acceptable salts and tautomers thereof, preparation method, pharmaceutical composition and application thereof

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and relates to a diketone nitrogen heterocyclic compound, a preparation method thereof, a pharmaceutical composition and application thereof, in particular to the diketone nitrogen heterocyclic compound or pharmaceutically acceptable salts and tautomers thereof, and the preparation method, the pharmaceutical composition and the application thereof.

Background

SARS-CoV-2 is a highly pathogenic, pandemic human and animal co-virus belonging to the coronaviridae family with SARS-CoV-1 and MERS-CoV. These three viruses, unlike the other several coronaviruses HCoV-NL63, HCoV-229E, HCoV-OC43 and HCoVHKU, can cause severe respiratory diseases. Symptoms of SARS-CoV-2 infection range from asymptomatic disease to moderate and severe pneumonia, as well as life threatening complications including hypoxic respiratory failure, acute respiratory distress syndrome, multiple system organ failure, and ultimately death. Even more terrible, the virus is not only highly contagious, but can be transmitted by asymptomatic infected persons and those in both symptomatic and pre-symptomatic stages. Although many different vaccines are currently approved for sale or emergency use worldwide, a significant portion of the population worldwide is not vaccinated due to limitations in its own physical or local medical conditions. In addition, vaccines have reduced protective efficacy against SARS-CoV-2 variants, particularly the recently-developed worldwide Omicron strain. Thus, the development of new crown drugs that are effective against variabilities is urgent.

Coronaviruses are broken down to release nucleocapsids and viral genomes after entering host cells. The host cell ribosomes translate the Open Reading Frames (ORFs) 1a and 1b of the viral genome into the multimeric proteins pp1a and pp1b, respectively, which encode 16 nonstructural proteins (nsps), while the remaining ORFs encode structural and accessory proteins. The cleavage of PP to nsp2-16 is catalyzed by 3C-like cysteine protease (3 CLpro) and papain-like cysteine protease (PLpro) to form the replication-transcription complex (RTC). These protease activity deletions lead to a viral life cycle arrest. Furthermore, the structure and function of 3CLpro are highly conserved among coronaviruses. 3CLpro catalytic center mutation rate is extremely low, and drug resistance is not easy to generate; the 3Clpro inhibitor should be effective against all variants, not dependent on inducing an immune response, but rather blocking the viral replication protease 3CLpro by binding to the viral backbone. The polypeptide after cleavage of only the glutamine (Gln) residue by 3CLpro, no known human protease has shown the same cleavage specificity as 3CLpro, and thus the potential toxicity of 3CLpro inhibitors is lower. Thus, 3CLpro is an effective target for the development of oral anti-neocrown drugs.

The 3CLpro inhibitors reported so far include covalent peptidomimetic inhibitors represented by PF-07321332 developed by the company pyroxene and non-covalent, non-peptidomimetic small molecule inhibitors represented by S-217622 developed by the company japanese Shiongai (salt field sense) pharmaceutical. At present, the new oral drug Paxlovid of the new crown of the xenia (the main component is PF-07321332) is obtained as an emergency use authorization of the FDA and becomes the first oral new crown drug obtained in the United states. Additional conditions of the Chinese drug administration approve Paxlovid import registrations for the treatment of light to moderate new patients with advanced high risk factors. PF-07321332 is a substrate for CYP3A4 and is metabolically unstable and must be taken together with the CYP3A4 enzyme inhibitor ritonavir. Changes in the activity of the CYP3A4 enzyme affect Paxlovid metabolism, which in turn affects Paxlovid effectiveness and safety. S-217622 is hopeful to get rid of dependence on P450 enzyme inhibitors (such as ritonavir), realizes a new crown of single drug treatment, expands the applicable crowd range without taking any trouble and other drugs needing to be taken simultaneously generate pharmacological reaction due to the inhibition effect of the P450 enzyme. Although S-217622 shows great potential for treating a new crown, the currently reported non-covalent small molecule inhibitors are still very deficient, and have the problems of single structure, weak enzyme inhibition activity, poor patent medicine and the like. Therefore, the searching of a novel, efficient and low-toxicity 3CLpro non-covalent small molecule inhibitor has important significance, and provides more and more practical clinical drug treatment options for new patients with different symptoms.

Disclosure of Invention

Aiming at the problems of single structure of a broad-spectrum antiviral drug and lack of a non-covalent high-efficiency 3CLpro small molecule inhibitor in the prior art, the invention provides a diketone nitrogen heterocyclic compound, a preparation method, a pharmaceutical composition and application thereof. The diketone nitrogen heterocyclic compound is a 3CLpro non-covalent small molecule inhibitor with remarkable activity, and has better therapeutic effect on coronavirus infectious diseases.

The invention discloses a diketone nitrogen heterocyclic compound with a structure shown in a general formula I or pharmaceutically acceptable salt, tautomer, metabolite, prodrug, solvate or hydrate thereof, which has the following structure:

wherein, R ¹ is hydrogen, deuterium, C _3-10 cycloalkyl, C _1-6 alkyl;

R ² is halogen, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 haloalkoxy or C _1-6 haloalkyl;

R ³ is halogen, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 haloalkoxy or C _1-6 haloalkyl;

L is-NR ₄ -, -NHCO-, -CONH-, or-NH-NH-;

R ⁴ is hydrogen, unsubstituted or R ^4-1 substituted C _1-4 alkyl;

r ^4-1 is cyano, amino or hydroxy;

A is

The a end is connected with L, and the b end is connected with LConnected with the c terminal/>Are connected;

when A is When L is-NR ⁴ -, R ⁴ is unsubstituted or R ^4-1 substituted C _1-4 alkyl.

Preferably, a diketone nitrogen heterocyclic compound with a structure shown in a general formula I or pharmaceutically acceptable salt, tautomer, metabolite, prodrug, solvate or hydrate thereof, wherein when R ¹ is C _3-10 cycloalkyl, the C _3-10 cycloalkyl is C _3-6 cycloalkyl;

And/or, when R ¹ is C _1-6 alkyl, said C _1-6 alkyl is C _1-4 alkyl;

And/or, when R ² is halogen, said halogen is fluorine, chlorine, bromine or iodine;

And/or, when R ² is C _1-6 alkyl, said C _1-6 alkyl is C _1-4 alkyl;

and/or, when R ² is C _1-6 alkoxy, said C _1-6 alkoxy is C _1-4 alkoxy;

And/or, when R ² is C _1-6 haloalkyl, said C _1-6 haloalkyl is C _1-4 haloalkyl;

And/or, when R ² is C _1-6 haloalkoxy, said C _1-6 haloalkoxy is C _1-4 haloalkoxy;

and/or, when R ³ is halogen, said halogen is fluorine, chlorine, bromine or iodine;

And/or, when R ³ is C _1-6 alkyl, said C _1-6 alkyl is C _1-4 alkyl;

And/or, when R ³ is C _1-6 alkoxy, said C _1-6 alkoxy is C _1-4 alkoxy;

and/or, when R ³ is C _1-6 haloalkyl, said C _1-6 haloalkyl is C _1-4 haloalkyl;

And/or, when R ³ is C _1-6 haloalkoxy, said C _1-6 haloalkoxy is C _1-4 haloalkoxy;

And/or, when R ⁴ is unsubstituted or R ^4-1 substituted C _1-4 alkyl, said C _1-4 alkyl is methyl, ethyl or propyl;

And/or, when R ⁴ is unsubstituted or R ^4-1 substituted C _1-4 cycloalkyl, the number of R ^4-1 is one or more, and when a plurality of R ^4-1 are present, the R ^4-1 may be the same or different.

Preferably, when R ¹ is C _3-10 cycloalkyl, said C _3-10 cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl;

And/or, when R ¹ is C _1-6 alkyl, said C _1-6 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl;

and/or, when R ² is C _1-6 alkyl, said C _1-6 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl;

And/or, when R ² is C _1-6 alkoxy, said C _1-6 alkoxy is methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy and tert-butoxy;

And/or, when R ² is C _1-6 haloalkyl, said C _1-6 haloalkyl is trifluoromethyl;

And/or, when R ² is C _1-6 haloalkoxy, said C _1-6 haloalkoxy is trifluoromethoxy;

and/or, when R ³ is C _1-6 alkyl, said C _1-6 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl;

and/or, when R ³ is C _1-6 alkoxy, said C _1-6 alkoxy is methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy and tert-butoxy;

and/or, when R ³ is C _1-6 haloalkyl, said C _1-6 haloalkyl is trifluoromethyl;

And/or, when R ³ is C _1-6 haloalkoxy, said C _1-6 haloalkoxy is trifluoromethoxy;

And/or, when R ⁴ is C _1-4 alkyl substituted by R ^4- 1, said C _1-4 alkyl substituted by R ^4-1 is

Preferably, when R ¹ is hydrogen, deuterium, or C _1-6 alkyl;

R ² is halogen;

R ³ is halogen;

l is-NR ⁴ -or-NH-NH-;

R ⁴ is hydrogen, unsubstituted or R ^4-1 substituted C _1-4 alkyl;

r ^4-1 is cyano or hydroxy;

A is

The a end is connected with L, and the b end is connected with LConnected with the c terminal/>Are connected.

Further, the compound shown in the formula I is any one of the following compounds:

the invention also discloses a preparation method of the diketone nitrogen heterocyclic compound with the structure shown in the general formula I or pharmaceutically acceptable salts, tautomers, metabolites, prodrugs, solvates or hydrates thereof:

The method comprises the following steps: in a solvent, the compound II and the compound III generate a compound I under the action of a base/condensing agent, a base/catalyst/ligand or a base;

Wherein X is amino, carboxyl, -NH-NH ₂ or-NHR ⁴, Y is halogen or C _1-3 alkylthio, Y is connected with the a end of A; r ¹、R²、R³、R⁴, L and A are as described above.

The conditions and operation of the reactions described above are the same as those conventional in the art for such reactions.

A pharmaceutical composition comprising a therapeutically effective amount of a diketone nitrogen heterocyclic compound having a structure represented by formula I or a pharmaceutically acceptable salt, tautomer, metabolite, prodrug, solvate, or hydrate thereof, and a pharmaceutically acceptable carrier or adjuvant.

The invention also discloses application of the diketone nitrogen heterocyclic compound with the structure shown in the general formula I or pharmaceutically acceptable salts, tautomers, metabolites, prodrugs, solvates or hydrates thereof, which is used for preparing the 3C-like cysteine protease inhibitor; or for the preparation of a medicament for the treatment and/or prophylaxis of viral infectious diseases.

The invention also discloses application of the pharmaceutical composition, which is used for preparing a 3C-like cysteine protease inhibitor; or for the preparation of a medicament for the treatment and/or prophylaxis of viral infectious diseases.

Further, the virus includes, but is not limited to, severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2), middle east respiratory syndrome-related coronavirus (MERS-CoV), severe acute respiratory syndrome-related coronavirus (SARS-CoV), influenza A virus, influenza B virus, spanish influenza virus, arenavirus, bunyavirus, rabies virus, avian influenza virus, bone marrow poliovirus, rhinovirus, adenovirus, ebola virus, enterovirus, hepatitis A virus, hepatitis C virus, hepatitis E virus, enterovirus, HIV virus, echovirus, filovirus, measles virus, yellow fever virus, japanese encephalitis virus, west Nile virus, newcastle disease virus, RS virus, vesicular stomatitis virus, mumps virus, dengue virus, coxsackievirus, rotavirus or tobacco mosaic virus.

The pharmaceutical excipients can be those which are widely used in the field of pharmaceutical production. Adjuvants are used primarily to provide a safe, stable and functional pharmaceutical composition, and may also provide means for allowing the subject to dissolve at a desired rate after administration, or for promoting effective absorption of the active ingredient after administration of the composition. The pharmaceutical excipients may be inert fillers or provide a function such as stabilizing the overall pH of the composition or preventing degradation of the active ingredients of the composition. The pharmaceutical excipients can comprise one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, sizing agents, disintegrants, lubricants, anti-adherents, glidants, wetting agents, gelling agents, absorption retarders, dissolution inhibitors, enhancing agents, adsorbents, buffering agents, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

The pharmaceutical compositions of the present invention may be prepared in accordance with the disclosure using any method known to those of skill in the art. For example, conventional mixing, dissolving, granulating, emulsifying, levigating, encapsulating, entrapping or lyophilizing processes.

The pharmaceutical compositions of the present invention may be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular). The pharmaceutical compositions of the invention may also be in controlled or delayed release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, caplets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops or serum formulations. Examples of formulations for parenteral administration include, but are not limited to, solutions for injection, dry formulations which may be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic formulations; aerosol: such as nasal sprays or inhalants; a liquid dosage form suitable for parenteral administration; suppositories and lozenges.

The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention prepared from the compounds of the present invention which have the specified substituents found herein with relatively non-toxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts may be obtained by contacting the free form of such compounds with a sufficient amount of base in pure solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic ammonia or magnesium salts or similar salts. When the compounds of the present invention contain relatively basic functional groups, the acid addition salts may be obtained by contacting the free form of such compounds with a sufficient amount of acid in pure solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming carbonates or bicarbonates), phosphoric acid (forming phosphates, monohydrogenphosphates, dihydrogenphosphates, sulfuric acid (forming sulfates or bisulphates), hydroiodic acid, phosphorous acid, and the like, and organic acid salts including, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like, salts of amino acids (such as arginine and the like), and salts of organic acids such as glucuronic acid.

The "pharmaceutically acceptable salts" of the present invention can be synthesized from the parent compound containing an acid or base by conventional chemical methods. In general, the preparation of such salts is as follows: prepared via reaction of these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both. Generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

The term "tautomer" refers to a functional group isomer that results from the rapid movement of an atom in a molecule at two positions, such as the interconversion between enamines and imines:

the term "metabolite" refers to a pharmaceutically active product of a compound of formula I or a salt thereof produced by in vivo metabolism. Such products may result from, for example, oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, glucuronidation, enzymatic cleavage, etc. of the administered compound. Accordingly, the present invention includes metabolites of the compounds of the present invention, including compounds produced by a method of contacting a compound of the present invention with a mammal for a period of time sufficient to obtain the metabolites thereof.

Identification of metabolites typically occurs by preparing a radiolabeled isotope of a compound of the invention, parenterally administering it to an animal, such as a rat, mouse, guinea pig, monkey or human, in a detectable dose (e.g., greater than about 0.5 mg/kg), allowing sufficient time for metabolism to occur (typically about 30 seconds to 30 hours) and isolating its conversion product from urine, blood or other biological samples. These products are easy to isolate because they are labeled (others are isolated by using antibodies that are capable of binding to epitopes present in the metabolite). The metabolite structures are determined in a conventional manner, for example by MS, LC/MS or NMR analysis. In general, the analysis of metabolites is performed in the same manner as conventional drug metabolism studies known to those skilled in the art. So long as the metabolite products are not otherwise undetectable in vivo, they are useful in assays for therapeutic dosing of the compounds of the invention. The compounds of the present invention may contain non-natural proportions of atomic isotopes on one or more of the atoms comprising the compounds. For example, compounds such as tritium (3H), iodine-125 (125I) or C-14 (14C) may be labeled with a radioisotope. All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

In addition to salt forms, the compounds provided herein exist in prodrug forms. Prodrugs of the compounds described herein readily undergo chemical changes under physiological conditions to convert to the compounds of the invention. Any compound that can be converted in vivo to provide a biologically active substance (i.e., a compound of formula I) is a prodrug within the scope and spirit of the invention. For example, compounds containing a carboxyl group can form a physiologically hydrolyzable ester that acts as a prodrug by hydrolyzing in vivo to give the compound of formula I itself. The prodrugs are preferably administered orally, as hydrolysis occurs in many cases primarily under the influence of digestive enzymes. Parenteral administration may be used when the ester itself is active or hydrolysis occurs in the blood.

Those skilled in the art will appreciate that, in accordance with the convention used in the art, the present application describes the structural formula of the group usedMeaning that the corresponding group is linked through this site to other fragments, groups in the compound of formula I.

"Substitution" in the present invention may be one or more, and when there are plural "substitution", the "substitution" may be the same or different.

The term "plurality" may enumerate, for example, 2,3, or 4.

The term "halogen" includes fluorine, chlorine, bromine or iodine.

The term "alkyl" refers to a straight or branched chain alkyl group having the indicated number of carbon atoms. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl and the like.

The term "alkoxy" refers to the group-O-RY, wherein RY is alkyl as defined above.

The term "cycloalkyl" refers to a saturated, monocyclic or polycyclic alkyl group. The monocyclic cycloalkyl group is preferably a monovalent saturated cyclic alkyl group having 3 to 7 ring carbon atoms, more preferably 3 to 6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. Each ring of the polycyclic cycloalkyl is saturated and may be a bicyclic or tricyclic cycloalkyl having 4 to 10 carbon atoms.

The above preferred conditions can be arbitrarily combined on the basis of not deviating from the common knowledge in the art, and thus, each preferred embodiment of the present invention can be obtained.

The reagents and materials used in the present invention are commercially available.

The beneficial effects are that: compared with the prior art, the invention has the following advantages:

(1) The diketone nitrogen heterocyclic compound has good inhibitory activity on 3C-like cysteine protease, and the minimum half inhibitory concentration of the compound can reach 0.009 mu M in an enzyme inhibitory activity test experiment and is far lower than that of a positive control 0.028 mu M;

(2) The diketone nitrogen heterocyclic compound has good treatment effect on infectious diseases, the inhibitory activity EC ₅₀ of the compound in Vero E6 cells on virus infection can reach 0.20 mu M at the lowest, the positive control is obviously better than 0.58 mu M, and the compound can inhibit the virus titer to the lowest detection limit in a virus infection mouse model;

(3) The diketone nitrogen heterocyclic compound has small toxic and side effects, and the cytotoxic IC ₅₀ of the diketone nitrogen heterocyclic compound on Vero E6 cells is more than 100 mu M.

Drawings

FIG. 1 is a graph showing anti-infective activity of positive control group and compound S11 in a mouse infection model in example 38 of the present invention;

FIG. 2 is a graph showing anti-infective activity of positive control group and compound S22 in a mouse infection model in example 38 of the present invention;

FIG. 3 is a graph showing the anti-infective activity of the positive control group and compound S31 in a mouse infection model in example 38 of the present invention.

Detailed Description

The invention is further described below with reference to the drawings and examples.

Example 1

Synthesis of compound S1:

Step one: synthesis of Compound 2

Compound 1 (15 g,98.7 mmol) was dissolved in anhydrous DMF (200 mL), DBU (15.8 g,103.6 mmol) was added at 0deg.C, then tert-butyl isocyanate (7) (10.3 g,103.6 mmol) was slowly added dropwise and after the addition was complete, reacted overnight at 0deg.C. The next day, DBU (18 g,118.4 mmol) and CDI (19.2 g,118.4 mmol) were added to the reaction solution, which was then transferred to room temperature and stirred for 6h. The reaction mixture was quenched by addition of dilute hydrochloric acid at 0deg.C, extracted with EA (100 mL. Times.3), washed with saturated brine (200 mL), dried over anhydrous Na ₂SO₄, filtered, concentrated, and purified by column chromatography (PE: EA=3:1) to give the compound 2(19g,80％).¹H NMR(300MHz,Chloroform-d)δ11.26(s,1H),4.27(q,J＝7.1Hz,2H),1.62(s,9H),1.37–1.27(m,3H).

Step two: synthesis of Compound 3

Compound 2 (19 g,78.8 mmol) was dissolved in acetonitrile (240 mL), and to the above solution were added compound 8 (26 g,118.8 mmol) and K ₂CO₃ (16.4 g,118.8 mol), and the reaction solution was heated under reflux for 3h. The reaction solution was cooled to room temperature, suction-filtered, the filtrate was concentrated, and the compound was obtained by column chromatography separation and purification (PE: ea=30:1) 3(25.8g,85％).¹H NMR(300MHz,Chloroform-d)δ7.30(dtt,J＝8.0,5.0,1.0Hz,1H),6.94(td,J＝8.1,5.0Hz,1H),5.10(s,2H),4.28(q,J＝7.1Hz,2H),1.37(s,9H),1.33(t,J＝7.1Hz,3H).

Step three: synthesis of Compound 4

Compound 3 (20 g,51.9 mmol) was dissolved in TFA (39 mL), stirred at room temperature for 6h, stirring stopped, TFA was distilled off under reduced pressure, ether was slurried, suction filtered, the filter cake was collected, and dried under vacuum to give the compound 4(15.4g,90％).¹H NMR(300MHz,Chloroform-d)δ9.22(s,1H),7.32(dtt,J＝8.1,5.0,1.0Hz,1H),6.94(td,J＝8.0,5.0Hz,1H),5.02(s,2H),4.32(q,J＝7.1Hz,2H),1.31(t,J＝7.1Hz,3H).

Step four: synthesis of Compound 5

Compound 4 (15 g,45.6 mmol) was dissolved in anhydrous DMF (80 mL), and to the above solution were added compound 9 (11.4 g,68.4 mmol) and K ₂CO₃ (18.9 g,136.8 mol), the reaction was warmed to 60℃and stirred for 4h. The reaction was cooled to room temperature, quenched with water (100 mL), extracted with DCM (100 ml×3), the combined organic phases washed with saturated brine (200 mL), dried over anhydrous Na ₂SO₄, filtered, concentrated, and purified by column chromatography (DCM: meoh=80:1) to give compound 5 (7.7 g, 40%).

Step five: synthesis of Compound 6

Compound 5 (7.7 g,18.2 mmol) was dissolved in methanol, aqueous NaOH (1M, 27.4mmol,27 mL) was added at 0deg.C, and the mixture was transferred to room temperature and reacted under stirring for 5h. Transferring the reaction solution to 0 ℃, regulating the pH value of a 2N HCl solution to 2-3, separating out solids, carrying out suction filtration, collecting a filter cake, and carrying out vacuum drying to obtain the compound 6, wherein the compound 6 is directly put into the next reaction without purification.

Step six: synthesis of Compound S1

Compound 6 (6.5 g,16.4 mmol) was dissolved in anhydrous DMF (30 mL), and to the above solution was added compound 10 (3.3 g,18 mmol), HATU (8.2 g,21.32 mmol) and DIPEA (8.6 mL,49.2 mmol) and the reaction was stirred at room temperature overnight. The reaction was stopped, quenched by adding water to the reaction mixture, extracted with EA (50 ml×3), the organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous Na ₂SO₄, filtered, concentrated, and purified by column chromatography (DCM: meoh=100:1) to give the compound S1(6.0g,65％).¹H NMR(500MHz,DMSO-d₆,DCl in D₂O)δ9.91(s,1H),8.23(s,1H),8.08(d,J＝1.5Hz,1H),7.67(d,J＝1.8Hz,1H),7.56(s,1H),7.06–6.91(m,2H),5.17(s,2H),5.08(s,2H),4.03(s,3H),3.95(s,3H).m/z(ESI-MS):560.1[M+H]⁺.

Example 2

Synthesis of Compound S2

The reaction process and reaction conditions of the first step are the same as those of the example 1, and the compound 1 is replaced by the corresponding raw material compound 11, and the second, third and fourth steps are performed according to the method in the example 1.

Step five: synthesis of Compound 15

Compound 15 (10 g,24.3 mmol) was placed in a closed tube, ammonia/methanol (7M, 17mL,121 mmol) was added, the reaction was warmed to 50℃and stirred overnight. Cooling to room temperature, concentrating under reduced pressure, pulping by diethyl ether, filtering, collecting filter cakes, and vacuum drying to obtain the compound 16, wherein the compound 16 is directly put into the next reaction without further purification.

Step six: synthesis of Compound S2

The reaction process and reaction conditions were the same as in example 1, except that compound 10 was replaced with the corresponding starting compound 17 .¹H NMR(500MHz,DMSO-d₆,DCl in D₂O)δ8.28–8.23(m,2H),7.77(d,J＝1.8Hz,1H),7.56(s,1H),7.50–7.42(m,1H),7.21(s,1H),6.99(td,J＝8.0,5.0Hz,1H),5.19(s,2H),5.08(s,2H),4.03(s,3H),3.95(s,3H).m/z(ESI-MS):560.1[M+H]+.

Example 3

Synthesis of Compound S3

Compound 18 (6755 mg,3 mmol) was dissolved in anhydrous THF (12 mL), nitrogen blanket, liHMDS (1M, 4.5mL,4.5 mmol) was added dropwise to the above solution at 0deg.C, then a THF solution of compound 15 (284 mg,2 mmol) was added dropwise slowly to the above solution, reacted at 0deg.C for 2h, and transferred to room temperature for 1h. Then, the reaction mixture was transferred to 0 ℃, quenched by addition of saturated ammonium chloride solution, extracted with EA (10 ml×3), washed with saturated brine (10 mL), dried over anhydrous Na ₂SO₄, filtered, concentrated, and purified by column chromatography (DCM: meoh=20:1) to give the compound S3(288mg,25％).¹H NMR(500MHz,500MHz,DMSO-d₆,DCl in D₂O)δ8.27(s,1H),8.00(d,J＝1.5Hz,1H),7.69(d,J＝1.4Hz,1H),7.46(s,1H),7.13(dtt,J＝8.0,5.0,1.0Hz,1H),6.96(td,J＝7.9,5.0Hz,1H),5.05(d,J＝1.1Hz,2H),4.68(d,J＝12.5Hz,1H),4.44(t,J＝7.3Hz,1H),4.37–4.26(m,3H),4.02(s,3H),3.94(s,3H),3.88(q,J＝6.9Hz,2H).m/z(ESI-MS):576.2[M+H]+.

The synthesis of the compounds S4 to S10 in examples 4 to 10 below is performed by referring to the synthesis method of example 3, and only the corresponding raw materials need to be replaced.

Example 4

Synthesis of Compound S4

¹H NMR(500MHz,500MHz,DMSO-d₆,DCl in D₂O)δ8.22(s,1H),8.00(d,J＝1.5Hz,1H),7.78(d,J＝1.5Hz,1H),7.46(s,1H),7.37(dtt,J＝8.0,5.0,1.0Hz,1H),6.96(td,J＝8.0,4.9Hz,1H),5.04(d,J＝1.1Hz,2H),4.68(d,J＝12.5Hz,1H),4.49(t,J＝5.5Hz,2H),4.30(d,J＝12.6Hz,1H),4.03(s,3H),3.96(s,3H),2.87(t,J＝5.5Hz,2H).m/z(ESI-MS):585.2[M+H]+.

Example 5

Synthesis of Compound S5

¹H NMR(500MHz,500MHz,DMSO-d₆,DCl in D₂O)δ8.23(s,1H),8.01(dd,J＝15.9,1.6Hz,2H),7.59(s,1H),7.38(dtt,J＝7.9,4.9,1.0Hz,1H),6.98(td,J＝8.0,5.0Hz,1H),5.05(d,J＝1.1Hz,2H),4.97(s,2H),4.68(d,J＝12.5Hz,1H),4.30(d,J＝12.6Hz,1H),4.03(s,3H),3.95(s,3H)..m/z(ESI-MS):571.2[M+H]+.

Example 6

Synthesis of Compound S6

¹H NMR(500MHz,500MHz,DMSO-d₆,DCl in D₂O)δ8.15(s,1H),7.98(d,J＝1.5Hz,1H),7.77(d,J＝1.7Hz,1H),7.45(dtt,J＝8.0,5.0,1.1Hz,1H),7.30(s,1H),6.91(td,J＝8.0,5.0Hz,1H),5.07(d,J＝0.9Hz,2H),4.68(d,J＝12.5Hz,1H),4.30(d,J＝12.6Hz,1H),4.03(s,3H),3.95(s,3H),3.73(s,3H).m/z(ESI-MS):546.2[M+H]+.

Example 7

Synthesis of Compound S7

¹H NMR(500MHz,500MHz,DMSO-d₆,DCl in D₂O)δ8.20(s,1H),8.02(s,1H),7.89(d,J＝1.8Hz,1H),7.53(d,J＝1.5Hz,1H),7.43(s,1H),7.18(dtt,J＝8.0,4.9,1.1Hz,1H),6.97–6.89(m,2H),4.99(d,J＝1.1Hz,2H),4.68(d,J＝12.5Hz,1H),4.30(d,J＝12.6Hz,1H),4.02(s,3H),3.95(s,3H).m/z(ESI-MS):547.2[M+H]+.

Example 8

Synthesis of Compound S8

¹H NMR(500MHz,500MHz,DMSO-d₆,DCl in D₂O)δ8.22(s,1H),8.03(s,1H),7.88(d,J＝1.5Hz,1H),7.57(dd,J＝5.0,1.5Hz,1H),7.51(tt,J＝5.0,1.0Hz,1H),7.00(d,J＝8.0Hz,1H),6.93(t,J＝8.0Hz,1H),6.19(d,J＝6.6Hz,1H),5.00(d,J＝1.1Hz,2H),4.68(d,J＝12.5Hz,1H),4.30(d,J＝12.6Hz,1H),4.02(s,3H),3.95(s,3H).m/z(ESI-MS):547.2[M+H]+.

Example 9

Synthesis of Compound S9

¹H NMR(500MHz,500MHz,DMSO-d₆,DCl in D₂O)δ8.16(s,1H),8.05(d,J＝1.4Hz,1H),7.75(d,J＝1.3Hz,1H),7.52(s,1H),7.20(dtd,J＝7.9,5.0,1.0Hz,1H),7.01(td,J＝8.0,5.0Hz,1H),6.41(qd,J＝6.2,1.0Hz,1H),5.02(s,1H),4.68(d,J＝1.1Hz,2H),4.03(s,3H),3.95(s,3H),1.57(d,J＝6.2Hz,3H).m/z(ESI-MS):546.2[M+H]+.

Example 10

Synthesis of Compound S10

¹H NMR(500MHz,500MHz,DMSO-d₆,DCl in D₂O)δ8.11(s,1H),8.06(d,J＝1.5Hz,1H),7.75(d,J＝1.4Hz,1H),7.52(s,1H),7.41(dtd,J＝8.1,5.1,1.0Hz,1H),6.94(td,J＝8.0,5.0Hz,1H),6.50(s,1H),5.34(dd,J＝6.0,1.1Hz,1H),4.69(d,J＝12.4Hz,1H),4.30(d,J＝12.6Hz,1H),4.03(s,3H),3.95(s,3H),1.76(q,J＝6.0Hz,1H),1.17–1.07(m,2H),0.87–0.78(m,2H).m/z(ESI-MS):572.1[M+H]+.

Example 11

Synthesis of Compound S11

Step one: synthesis of Compound 20

Compound 19 (5 g,34.5 mmol) and LiBr (3 g,34.5 mmol) were dissolved in anhydrous DMF (100 mL), naH (60%, 1.7g,41.4 mmol) was added in portions to the above solution at 0deg.C under nitrogen protection, and the reaction was stirred at 0deg.C for 1h. Then, a solution of Compound 8 (8.5 g,38 mmol) in DMF was slowly added dropwise to the above suspension, and the reaction mixture was allowed to reach room temperature and stirred for 30min. Then, the reaction mixture was transferred to 0℃and quenched with dilute hydrochloric acid, extracted with EA (50 mL. Times.3), washed with saturated brine (50 mL), dried over anhydrous Na ₂SO₄, filtered, concentrated, and purified by column chromatography (PE: EA=3:1) to give the compound 20(2g,20％).¹H NMR(300MHz,Chloroform-d)δ9.22(s,1H),7.18–7.10(m,1H),6.97(td,J＝8.1,5.0Hz,1H),5.69(s,1H),4.97(s,2H).

Step two: synthesis of Compound 21

Compound 20 (2 g,6.90 mmol) was dissolved in anhydrous DMF (20 mL), and to the above solution were added compound 9 (1.7 g,10.4 mmol) and K ₂CO₃ (1.4 g,10.4 mol), the reaction was warmed to 60℃and stirred for 4h. The reaction was cooled to room temperature, quenched with water (20 mL), extracted with DCM (20 ml×3), the combined organic phases washed with saturated brine (20 mL), dried over anhydrous Na2SO4, filtered, concentrated, and purified by column chromatography (DCM: meoh=50:1) to give the compound 21(1.2g,45％).¹H NMR(500MHz,Chloroform-d)δ8.27(s,1H),7.13(dtt,J＝8.0,4.9,1.0Hz,1H),6.96(td,J＝8.0,5.0Hz,1H),5.67(s,1H),4.97(s,2H),4.63(s,2H),4.06(s,3H).

Step three: synthesis of Compound S11

Compound 21 (803 mg,0.943 mmol) was dissolved in anhydrous 1, 4-dioxane (5 mL), and to the above solution was added compound 10 (mg, 1.41 mmol), pd (OAc) ₂ (21 mg,0.094 mmol), xantphos (82.0 mg,0.141 mmol) and CsCO ₃ (430 mg,1.32 mmol), and the reaction was heated under reflux for 1h. Cooled to room temperature, concentrated under reduced pressure, and purified by column chromatography (DCM: meoh=30:1) to give the compound S11(425mg,85％).¹H NMR(500MHz,DMSO-d₆)δ9.32(s,1H),8.52(d,J＝1.4Hz,1H),8.21(s,1H),8.01(d,J＝1.7Hz,1H),7.52(s,1H),7.35(dtt,J＝8.0,5.1,1.0Hz,1H),6.98(td,J＝7.9,5.0Hz,1H),5.17(s,2H),5.01(d,J＝1.1Hz,2H),4.64(s,1H),4.02(s,3H),3.93(s,3H).m/z(ESI-MS):531.1[M+H]+.

The synthesis of the compounds S12 to S21 in examples 12 to 21 below was performed by referring to the synthesis method of example 11, and only the corresponding raw materials were replaced.

Example 12

Synthesis of Compound S12

¹H NMR(500MHz,DMSO-d₆)δ8.50(d,J＝1.6Hz,1H),8.23(s,1H),8.16(s,1H),7.83(d,J＝1.5Hz,1H),7.27–7.18(m,2H),6.97(td,J＝8.0,4.9Hz,1H),5.13(s,2H),5.01(d,J＝0.9Hz,2H),4.62(s,1H),4.03(s,3H),3.96(s,3H).m/z(ESI-MS):581.2[M+H]+.

Example 13

Synthesis of Compound S13

¹H NMR(500MHz,DMSO-d₆)δ9.07(s,1H),8.38(dd,J＝5.1,1.5Hz,1H),8.21(s,1H),8.01(d,J＝1.6Hz,1H),7.61(tt,J＝5.0,1.0Hz,1H),7.17(d,J＝8.1Hz,1H),6.96(t,J＝8.0Hz,1H),5.16(s,2H),5.01(d,J＝0.9Hz,2H),4.64(s,1H),4.02(s,3H),3.95(s,3H).m/z(ESI-MS):531.2[M+H]+.

Example 14

Synthesis of Compound S14

¹H NMR(500MHz,DMSO-d₆)δ8.80(s,1H),8.52(d,J＝1.4Hz,1H),8.17(s,1H),8.01(d,J＝1.7Hz,1H),7.51(s,1H),7.25–7.18(m,1H),6.74(t,J＝8.0Hz,1H),5.17(s,2H),4.94(d,J＝0.9Hz,2H),4.61(s,1H),4.02(s,3H),3.92(s,3H),2.25(s,3H).m/z(ESI-MS):527.2[M+H]+.

Example 15

Synthesis of Compound S15

¹H NMR(500MHz,DMSO-d₆)δ9.08(s,1H),8.62(d,J＝1.4Hz,1H),8.20(s,1H),7.97(d,J＝1.5Hz,1H),7.67(s,1H),7.32(dt,J＝4.9,1.0Hz,1H),7.25(d,J＝8.1Hz,1H),5.20(s,2H),4.97(d,J＝1.1Hz,2H),4.62(s,1H),4.03(s,3H),3.95(s,3H).m/z(ESI-MS):597.2[M+H]+.

Example 16

Synthesis of Compound S16

¹H NMR(500MHz,DMSO-d₆)δ9.34(s,1H),8.17(s,1H),7.98(d,J＝1.8Hz,1H),7.51(s,1H),7.20–7.12(m,2H),7.00(td,J＝8.1,5.0Hz,1H),6.36(qd,J＝6.2,1.0Hz,1H),5.20(s,1H),4.70–4.60(m,2H),4.03(s,1H),3.94(s,3H),1.51(d,J＝6.2Hz,3H).m/z(ESI-MS):545.2[M+H]+.

Example 17

Synthesis of Compound S17

¹H NMR(500MHz,DMSO-d₆)δ8.17(s,1H),7.98(d,J＝1.8Hz,1H),7.52(s,1H),7.37(m,2H),7.04(d,J＝1.5Hz,1H),6.97(td,J＝8.0,4.9Hz,1H),5.17(s,1H),4.63(d,J＝2.5Hz,2H),4.03(s,1H),3.95(s,3H),1.88(h,J＝6.0Hz,1H),1.17–1.07(m,2H),0.81–0.72(m,2H)..m/z(ESI-MS):571.2[M+H]+.

Example 18

Synthesis of Compound S18

¹H NMR(500MHz,DMSO-d₆)δ8.17(s,1H),7.98(d,J＝1.8Hz,1H),7.52(s,1H),7.37(m,2H),7.04(d,J＝1.5Hz,1H),6.97(td,J＝8.0,4.9Hz,1H),5.17(s,1H),4.63(d,J＝2.5Hz,2H),4.03(s,1H),3.95(s,3H),1.88(h,J＝6.0Hz,1H),1.17–1.07(m,2H),0.81–0.72(m,2H)..m/z(ESI-MS):607.2[M+H]+.

Example 19

Synthesis of Compound S19

¹H NMR(500MHz,DMSO-d₆)δ8.23(s,1H),7.99(d,J＝1.5Hz,1H),7.87(d,J＝1.4Hz,1H),7.47(s,1H),7.27(d,J＝8.0Hz,1H),7.10(dt,J＝5.0,1.0Hz,1H),6.21(s,1H),5.11(s,2H),5.04(d,J＝1.1Hz,2H),4.32(t,J＝7.3Hz,1H),4.13(t,J＝6.8Hz,2H),4.03(s,3H),3.94(s,3H),3.87(q,J＝6.9Hz,2H).m/z(ESI-MS):607.2[M+H]+.

Example 20

Synthesis of Compound S20

¹H NMR(500MHz,DMSO-d₆)δ8.59(d,J＝6.6Hz,1H),8.29(s,1H),7.89(d,J＝1.8Hz,1H),7.63(d,J＝1.5Hz,1H),7.48–7.39(m,2H),6.98(td,J＝8.1,5.0Hz,1H),6.49(d,J＝6.6Hz,1H),5.35(s,2H),4.94(d,J＝0.9Hz,2H),4.62(s,1H),4.03(s,3H),3.95(s,3H).m/z(ESI-MS):546.2[M+H]+.

Example 21

Synthesis of Compound S21

¹H NMR(500MHz,DMSO-d₆)δ8.58(d,J＝6.6Hz,1H),8.27(s,1H),7.90(d,J＝1.6Hz,1H),7.68(s,1H),7.58(d,J＝1.7Hz,1H),7.03(dtt,J＝8.1,5.0,1.0Hz,1H),6.96(td,J＝8.1,5.0Hz,1H),5.80(s,2H),5.35(s,2H),4.96(d,J＝0.9Hz,1H),4.62(s,1H),4.03(s,3H),3.95(s,3H).m/z(ESI-MS):580.2[M+H]+.

Example 22

Synthesis of Compound S22

Step one: synthesis of Compound 23

Compound 22 (11.2 mL,98.4 mmol) was dissolved in anhydrous THF (100 mL), LDA (1M, 1mL,98.4 mmol) was slowly added dropwise to the solution at-78deg.C under nitrogen, and after the addition was completed, the reaction was continued with stirring for 30min. Then, compound 8 (26.3 g,118.1 mmol) was added dropwise to the above solution, and the mixture was transferred to room temperature and reacted under stirring for 3 hours. The reaction was transferred to 0deg.C, quenched by addition of saturated ammonium chloride solution, extracted with DCM (20 mL. Times.3), the combined organic phases washed with saturated brine (20 mL), dried over anhydrous Na ₂SO₄, filtered, concentrated, and dried in vacuo to give compound 23 which was directly taken to the next reaction without purification.

Step two: synthesis of Compound 24

Urea 27 (4.3 g,72.5 mmol) was dissolved in anhydrous methanol (150 ml), a methanol solution of compound 23 (20 g,72.5 mmol) was slowly added dropwise to the above solution at 0℃under nitrogen protection, and after the addition, the reaction solution was heated under reflux and stirred for 24 hours. After the reaction was completed, hot water and diluted hydrochloric acid were added to the above solution, and then the reaction solution was transferred to 0 ℃ and stirred overnight. The next day, suction filtration, ice water washing, filter cake collection and vacuum drying are carried out to obtain the compound 24(11.04g,56％).¹H NMR(300MHz,DMSO-d₆)δ8.13(s,2H),7.11(dtt,J＝8.1,5.0,1.0Hz,1H),6.93(td,J＝8.0,5.0Hz,1H),3.90(t,J＝8.5Hz,1H),3.42(dd,J＝8.5,1.1Hz,2H).

Step three: synthesis of Compound 25

Compound 24 (11 g,40.6 mmol) was dissolved in anhydrous DMF (80 mL), K ₂CO₃ (8.4 g,60.9 mmol) and compound 9 (7.5 g,44.7 mmol) were added to the above solution, the reaction was heated to 50deg.C and stirred for 4h. Cooled to room temperature, quenched with water, extracted with EA (50 ml×3), washed with saturated brine (50 mL), dried over anhydrous Na ₂SO₄, filtered, concentrated, and purified by column chromatography (DCM: meoh=40:1) to give the compound 25(6.7g,45％).¹H NMR(300MHz,DMSO-d₆)δ8.21(d,J＝8.4Hz,2H),7.01(dtt,J＝8.0,4.9,1.0Hz,1H),6.87(td,J＝8.0,5.0Hz,1H),5.02(d,J＝12.4Hz,2H),4.86(d,J＝12.5Hz,1H),4.01(s,2H),3.85(t,J＝8.5Hz,1H),3.46(ddd,J＝13.9,8.4,0.9Hz,1H),3.26(ddd,J＝14.1,8.5,1.1Hz,1H).

Step four: synthesis of Compound 26

Compound 25 (6.7 g,18.3 mmol) and BTAC (365 mg,3.66 mmol) were suspended in POCl ₃ (4.3 mL,45.8 mmol), the reaction was warmed to 50℃under nitrogen and stirred overnight. Cooled to room temperature and concentrated under reduced pressure. Then, ice sludges were slowly added to the resulting residue at 0 ℃, and the final slurry liquid was left to stand at 0 ℃ for 8 hours. Suction filtering, washing with ice water, collecting filter cake, vacuum drying to obtain compound 26(3.5g,50％).¹H NMR(300MHz,DMSO-d₆)δ8.26(s,2H),7.20(dtt,J＝8.0,5.0,1.0Hz,1H),6.95(td,J＝8.0,5.0Hz,1H),4.63(s,2H),4.06(s,2H),3.85(d,J＝0.9Hz,3H).

Step five: synthesis of Compound S22

Compound 26 (803 mg,0.943 mmol) was dissolved in anhydrous 1, 4-dioxane (5 mL), and to the above solution was added compound 10 (mg, 1.41 mmol), pd (OAc) ₂ (21 mg,0.094 mmol), xantphos (82.0 mg,0.141 mmol) and CsCO ₃ (430 mg,1.32 mmol), and the reaction was heated under reflux for 1h. Cooled to room temperature, concentrated under reduced pressure, and purified by column chromatography (DCM: meoh=30:1) to give the compound S22(375mg,75％).¹H NMR(500MHz,DMSO-d₆)δ8.55(d,J＝1.4Hz,2H),8.17(s,1H),8.01(d,J＝1.7Hz,1H),7.53(s,1H),7.22(dtt,J＝8.0,4.9,1.0Hz,2H),6.97(td,J＝8.0,5.0Hz,1H),4.61(s,2H),4.02(s,2H),3.93(s,3H),3.78(d,J＝1.1Hz,3H).m/z(ESI-MS):531.1[M+H]+.

The synthesis of the compounds S23 to S30 in examples 23 to 30 below was performed by referring to the synthesis method of example 22, and only the corresponding raw materials were replaced.

Example 23

Synthesis of Compound S23

¹H NMR(500MHz,DMSO-d₆)δ8.57(dd,J＝4.9,1.6Hz,2H),8.17(s,1H),8.01(d,J＝1.8Hz,1H),7.55(tt,J＝5.0,1.0Hz,1H),7.17(d,J＝8.1Hz,1H),6.98(t,J＝8.0Hz,2H),4.61(s,2H),4.02(s,3H),3.95(s,3H),3.79(d,J＝1.1Hz,2H).m/z(ESI-MS):531.2[M+H]+.

Example 24

Synthesis of Compound S24

¹H NMR(500MHz,DMSO-d₆)δ8.57(dd,J＝4.9,1.6Hz,1H),8.22(s,1H),8.01(d,J＝1.8Hz,1H),7.29–7.20(m,3H),7.17(d,J＝8.1Hz,2H),4.25(s,2H),4.02(s,3H),3.94(s,3H),3.68(d,J＝1.1Hz,2H).m/z(ESI-MS):531.2[M+H]+.

Example 25

Synthesis of Compound S25

¹H NMR(500MHz,DMSO-d₆)δ8.64(d,J＝1.4Hz,1H),8.20(s,1H),7.97(d,J＝1.5Hz,1H),7.67(s,1H),7.21(dtt,J＝8.0,4.9,1.0Hz,2H),6.98(td,J＝8.0,5.0Hz,2H),4.61(s,2H),4.03(s,2H),3.95(s,3H),3.74(d,J＝0.9Hz,3H).m/z(ESI-MS):565.2[M+H]+.

Example 26

Synthesis of Compound S26

¹H NMR(500MHz,DMSO-d₆)δ9.95(d,J＝6.6Hz,2H),9.73(s,1H),8.17(s,1H),7.89(d,J＝1.8Hz,1H),7.53(d,J＝1.7Hz,1H),7.46(s,1H),7.33(dtd,J＝7.9,5.0,1.0Hz,1H),6.93(td,J＝7.9,5.0Hz,1H),6.74(d,J＝6.6Hz,1H),4.31–4.21(m,3H),4.02(s,2H),3.95(s,3H),1.51(d,J＝7.0Hz,3H).m/z(ESI-MS):560.2[M+H]+.

Example 27

Synthesis of Compound S27

¹H NMR(500MHz,DMSO-d₆)δ8.16(s,1H),7.89(d,J＝1.8Hz,1H),7.53(d,J＝1.7Hz,1H),7.46(s,1H),7.23(dtt,J＝8.0,5.1,1.0Hz,1H),6.98(td,J＝8.1,5.0Hz,2H),6.83(d,J＝6.6Hz,2H),4.25(s,2H),4.03(s,3H),3.95(s,3H),3.69(d,J＝0.9Hz,2H).m/z(ESI-MS):546.2[M+H]+.

Example 28

Synthesis of Compound S28

¹H NMR(500MHz,DMSO-d₆)δ9.85(s,1H),8.22(s,1H),7.99(d,J＝1.5Hz,1H),7.85(d,J＝1.4Hz,1H),7.47(s,1H),7.06(dtt,J＝8.0,4.9,1.0Hz,1H),6.98(td,J＝8.1,5.0Hz,1H),4.32(t,J＝7.3Hz,2H),4.26(s,2H),4.17(t,J＝6.8Hz,2H),4.03(s,2H),3.94(s,3H),3.85(q,J＝6.9Hz,2H),3.78(d,J＝1.1Hz,2H).m/z(ESI-MS):575.2[M+H]+.

Example 29

Synthesis of Compound S29

¹H NMR(500MHz,DMSO-d₆)δ8.55(d,J＝1.5Hz,1H),8.17(s,1H),7.98(d,J＝1.7Hz,1H),7.53(s,1H),7.33(dtd,J＝7.9,4.9,1.0Hz,2H),6.99(td,J＝8.1,5.0Hz,2H),4.62(d,J＝2.9Hz,2H),4.45(qd,J＝6.9,1.0Hz,1H),4.03(s,2H),3.94(s,3H),1.48(d,J＝6.8Hz,3H).m/z(ESI-MS):545.2[M+H]+.

Example 30

Synthesis of Compound S30

¹H NMR(500MHz,DMSO-d₆)δ8.53(d,J＝1.5Hz,1H),8.18(s,1H),7.98(d,J＝1.5Hz,1H),7.51(s,1H),7.39(dtd,J＝8.1,5.1,1.1Hz,2H),6.98(td,J＝8.0,5.0Hz,2H),4.25(s,2H),4.03(s,2H),3.94(s,3H),3.58(dd,J＝6.7,1.0Hz,1H),1.96(dt,J＝6.7,5.9Hz,1H),1.26–1.12(m,5H).m/z(ESI-MS):571.2[M+H]+.

Example 31

Synthesis of Compound S31

Step one: synthesis of Compound 28

Compound 27 (14.5 g,100 mmol) was dissolved in anhydrous DMF (200 mL), csCO ₃ (49 g,150 mmol) and compound 8 (24.8 g,110 mmol) were added to the above solution, the reaction was warmed to 50deg.C and stirred for 5h. After the reaction solution was cooled to room temperature, quenched with water, extracted with DCM (200 ml×3), the organic phases were combined, washed with saturated brine (200 mL), dried over anhydrous Na ₂SO₄, filtered, concentrated, and purified by column chromatography (PE: ea=3:1) to give the compound 28(13.1g,45％).¹H NMR(500MHz,Chloroform-d)δ7.10(dtt,J＝8.1,5.0,1.0Hz,1H),6.96(td,J＝8.0,5.0Hz,1H),5.37(d,J＝0.9Hz,2H),3.77(s,2H).

Step two: synthesis of Compound 29

Compound 28 (10 g,34.5 mmol) was dissolved in anhydrous DMF (90 mL), naH (1.5 g,37.9 mmol) was added in portions to the above solution at 0deg.C, the reaction was stirred at 0deg.C for 1h, then a solution of compound 9 (6.3 g,37.9 mmol) in DMF was slowly added dropwise to the above solution, after the dropwise addition, the reaction solution was transferred to room temperature and stirred for 6h. Stopping the reaction, transferring the reaction solution to 0deg.C, quenching the reaction by adding saturated ammonium chloride, extracting with DCM (100 mL. Times.3), combining the organic phases, washing with saturated saline (100 mL), drying over anhydrous Na ₂SO₄, filtering, concentrating, and separating and purifying by column chromatography (DCM: meOH=30:1) to obtain the compound 29(8.6g,65％).1H NMR(500MHz,DMSO-d₆)δ8.23(s,1H),7.28(dtt,J＝8.0,4.9,1.0Hz,1H),6.96(td,J＝8.0,5.0Hz,1H),5.21(dd,J＝13.4,1.1Hz,1H),5.00(dd,J＝13.4,0.9Hz,1H),4.08(s,2H),3.68(t,J＝8.4Hz,2H),3.34(dd,J＝13.2,8.4Hz,1H),3.18(dd,J＝13.2,8.4Hz,1H).

Step three: synthesis of Compound S31

Compound 29 (803 mg,0.943 mmol) was dissolved in anhydrous 1, 4-dioxane (5 mL), and to the above solution was added compound 10 (mg, 1.41 mmol), pd (OAc) ₂ (21 mg,0.094 mmol), xantphos (82.0 mg,0.141 mmol) and CsCO ₃ (430 mg,1.32 mmol), and the reaction was heated under reflux for 1h. Cooled to room temperature, concentrated under reduced pressure, and purified by column chromatography (DCM: meoh=30:1) to give the compound S31(425mg,85％).1H NMR(500MHz,DMSO-d6)δ8.24(s,1H),8.01(dd,J＝20.3,1.5Hz,2H),7.54(s,2H),7.27–7.19(m,2H),6.97(td,J＝8.0,5.0Hz,1H),5.19(dd,J＝13.4,1.1Hz,1H),5.05(dd,J＝13.4,0.9Hz,1H),4.11–4.03(m,3H),3.93(s,3H),3.38(dd,J＝13.2,8.6Hz,1H),3.24(dd,J＝13.1,8.5Hz,1H).m/z(ESI-MS):531.1[M+H]+.

The synthesis of the compounds S32 to S35 in examples 32 to 35 below was performed by referring to the synthesis method of example 31, and only the corresponding raw materials were replaced.

Example 32

Synthesis of Compound S32

¹H NMR(500MHz,DMSO-d₆)δ8.42(s,1H),8.01(d,J＝1.5Hz,1H),7.91(d,J＝1.8Hz,1H),7.48(s,1H),7.29(dtt,J＝8.0,5.1,1.0Hz,1H),6.98(td,J＝8.1,5.0Hz,1H),5.20(dd,J＝13.4,0.9Hz,1H),5.01(dd,J＝13.4,1.1Hz,2H),4.60(t,J＝7.3Hz,1H),4.44(dt,J＝12.6,6.8Hz,1H),4.24(dt,J＝12.6,6.8Hz,1H),4.06(s,2H),3.95(s,3H),4.00–3.81(m,3H),3.46(dd,J＝13.2,8.4Hz,1H),3.29(dd,J＝13.2,8.4Hz,1H)..m/z(ESI-MS):575.2[M+H]+.

Example 33

Synthesis of Compound S33

¹H NMR(500MHz,DMSO-d₆)δ8.42(s,1H),8.01(d,J＝1.5Hz,1H),7.91(d,J＝1.8Hz,1H),7.48(s,1H),7.29(dtt,J＝8.0,5.1,1.0Hz,1H),6.98(td,J＝8.1,5.0Hz,1H),5.20(dd,J＝13.4,0.9Hz,1H),5.01(s,2H),4.06(s,2H),3.95(s,3H),4.00–3.81(m,3H),3.46(dd,J＝13.2,8.4Hz,1H),3.29(dd,J＝13.2,8.4Hz,1H).m/z(ESI-MS):546.2[M+H]+.

Example 34

Synthesis of Compound S34

¹H NMR(500MHz,DMSO-d₆)δ8.24(s,2H),8.06(d,J＝1.6Hz,1H),7.98(d,J＝1.6Hz,1H),7.52(s,2H),7.12(dtd,J＝7.9,5.0,1.0Hz,1H),6.98(td,J＝8.0,5.0Hz,1H),5.97(qd,J＝6.2,1.0Hz,1H),4.15–4.05(m,3H),3.95(s,3H),3.46(dd,J＝13.2,8.6Hz,1H),3.25(dd,J＝13.1,8.5Hz,1H),1.47(d,J＝6.2Hz,3H).m/z(ESI-MS):545.2[M+H]+.

Example 35

Synthesis of Compound S35

¹H NMR(500MHz,DMSO-d₆)δ8.25(s,2H),8.06(d,J＝1.6Hz,1H),7.98(d,J＝1.6Hz,1H),7.52(s,1H),7.17(dtd,J＝7.9,4.9,1.0Hz,2H),6.97(td,J＝8.0,5.0Hz,1H),5.58(dd,J＝6.0,0.9Hz,1H),4.14–4.04(m,3H),3.95(s,3H),3.41(dd,J＝13.1,8.5Hz,1H),3.24(dd,J＝13.1,8.5Hz,1H),1.90(p,J＝6.0Hz,1H),1.12(ddd,J＝10.5,9.8,5.8Hz,2H),0.81(ddd,J＝10.1,9.6,5.7Hz,2H).m/z(ESI-MS):571.2[M+H]+.

Example 36

Test of SARS-CoV-2 Virus 3C-like cysteine protease (3 CLpro) enzyme inhibitory Activity

1.3 Expression and purification of CLpro protein

The gene sequence of the full-length 3CLpro protein was constructed in the expression vector pET28a (+) vector and transferred into competent cells of E.coli BL21 (DE 3), and after induction for 12 hours at a final concentration of 0.5mM IPTG at 25℃was purified using a Ni-NTA column. The purified protein is detected by SDS, the part with the purity of more than 90 percent is further purified by Superdex 200 10/300GL of an AKTA Pure of a GE protein chromatography purification system, the protein with the purity of more than 95 percent is obtained, the protein concentration is measured by Nano Drop, and the protein is packaged and quick frozen by liquid nitrogen and then is stored at the temperature of minus 80 ℃.

Establishment of SARS-CoV-2 3CLpro enzyme activity screening system and calculation of inhibitor inhibition rate and medicine IC50

The SARS-CoV-2 3CLpro activity and the inhibitory activity of the compound on SARS-CoV-2 3CLpro are determined by Fluorescence Resonance Energy Transfer (FRET) technique. A fluorogenic substrate (Dabcyl-KTSAVLQ ∈ SGFRKM-E (Edans) -NH 2) with SARS-CoV-2 3CLpro cleavage site (arrow) and Tris-HCl buffer (20 mM Tris-HCl,150mM NaCl,10mM EDTA,pH 7.5) were used in the assay. The compound was dissolved in 100% dmso. Mu.l of the compound was incubated with 40. Mu.l of SARS-CoV-2 3CLpro (final concentration 0.5. Mu.M, tris-HCl buffer) at 25℃for 10min, and the reaction was initiated by addition of 50. Mu.l of fluorogenic substrate (final concentration 20. Mu.M). The Dabcyl fluorescent signal resulting from the cleavage of the substrate catalyzed by 3CLpro was detected using a radioresonance energy transfer fluorescence spectrophotometer at an excitation wavelength of 340nm and an absorption wavelength of 490 nm. SARS-CoV-2 3clpro kinetic constants (Vmax and Km) were obtained by fitting the data to MICHAELIS MENTEN equation, v=vmax× [ S ]/(km+ [ S ]). Kcat is then calculated according to the formula kcat=vmax/[ E ]. Compounds were diluted in a gradient by fold dilution using Tris-HCl buffer and assayed using the same final concentration of SARS-CoV-2 3CLpro and fluorogenic substrate system as described above. Values of intrinsic (V0 i) and apparent (Vappi, kappi) catalytic parameters of 3CLpro catalytic polypeptide substrate hydrolysis were determined in the presence and absence, respectively, of the compound of interest. The apparent inhibition constant (Kappi) for the binding of the target compound to Mpro is derived from the dependence of Vappi on the inhibitor concentration ([ I ]) at a fixed substrate concentration ([ S ]) according to the equation Vappi =Vappx [ I ]/(Kappi + [ I ]). The value of the intrinsic inhibition constant (Ki) for the binding of the target compound to 3CLpro is calculated according to equation Kappi = ki× (1+ [ S ]/Km). Inhibition curves for compounds were plotted by GRAPHPAD PRISM 8.0.0 software and IC50 values calculated. The following activities are superior to those shown in Table 1, and the example S-2 was conducted to compound 17 and 62. Has better effect on SARS-CoV-2 virus 3CLpro

TABLE 1 SARS-CoV-2 Virus 3CLpro enzyme inhibitory Activity

Example 37

Cytotoxicity and anti-SARS-CoV-2 virus infection efficacy test experiment

Vero E6 cytotoxicity test: the CCK8 method is used for detecting cytotoxicity of the compound to be detected in Vero E6 cells of mammals. Vero E6 cells were added to 96-well plates and cultured overnight. Cells were then incubated with different concentrations of test compound for 48h. The medium in the well plate was removed, replaced with fresh serum-free medium, 10% cck8 reagent was added and incubated at 37 ℃ for 1h, followed by detection of absorbance at 450nm using an enzyme-labeled instrument.

Screening compounds with no cytotoxicity or less cytotoxicity for testing antiviral infection, the specific operation comprises the following steps:

① Inoculating cells: taking Vero-E6 cells in logarithmic growth phase, sucking out the culture solution, and digesting the cells with pancreatin, wherein the cell count is as follows: 1X 106/mL; taking 4mL of the above cells, adding 6mL of culture medium, preparing a cell suspension with the cell density of 4X 105 cells/mL, inoculating into a 96-well plate, and inoculating 100 μl of the cell suspension into 4X 10 ⁴ cells per well. ② Drug pretreatment cells: the cell culture medium was replaced with DMEM medium containing 2% fbs, and 100 μl of the corresponding concentration of drug and DMSO was added to each well, followed by pretreatment in a 37 ℃ incubator for 1 hour. ③ Viral infection: taking 0.3mL of virus, adding 45mL of culture medium, uniformly mixing, and diluting the virus to 100TCID50/0.05mL; discarding the medicine culture medium vertical hanging drop virus diluent in the cell plate, adding 50 μl/hole of sample volume, adding corresponding medicine culture medium (containing medicine with corresponding concentration), adding 50 μl/hole of sample volume, and mixing; ④ incubation: the cell culture plates with the added samples are evenly mixed on a shaker and placed in a 37 ℃ incubator for incubation for 1h. After the incubation, the virus-serum mixed solution inoculated with cells is sucked, the medicines with corresponding concentration and the control group DMSO are added, the sample adding volume is 100 μl/hole (100 TCID 50/hole), and the mixture is placed in a CO ₂ incubator at 37 ℃ for culturing for 48 hours; ⑤ The supernatant was collected to detect viral RNA and immunofluorescent staining was performed with 4% paraformaldehyde fixing staining.

The specific experimental results are shown in Table 2, the compound of the example has smaller cytotoxicity, better inhibition activity on SARS-CoV-2 virus infection, better than positive control S-217662 and better selection index.

TABLE 2 cytotoxicity and anti-SARS-CoV-2 Virus infection Activity of test Compounds

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Example 38

In vivo anti-infective Activity test of Compounds S11, S22 and S31

Female BALB/c mice were first anesthetized by intraperitoneal injection of ketamine/xylazine (50 mg/kg/5 mg/kg), then SARS-CoV-2 gamma strain (1X 10 ⁴TCID₅₀ /) was inoculated intranasally to construct an infection model, and the negative control mice were instilled with the same volume of physiological saline. After successful molding, the blank control group, the S-217622 positive control group and the administration group are divided into 6 groups. Compounds S-217622 and S11 were each suspended in 0.5% methylcellulose and administered once orally immediately after molding was successful, and once after 12 h. S11 was administered at 2mg/kg,8mg/kg,16mg/kg and 32mg/kg, and S-217622 was administered at 32mg/kg. Mice were observed for pulmonary viral titers 24h after viral infection.

As shown in fig. 1,2 and 3, compounds S11, S22 and S31 significantly reduced viral titers in lung homogenates of infected mice relative to the placebo group and were dose dependent after two administrations. Positive control S-217622 and compounds S11, S22 and S31 reached the lowest limit of detection of viral titers at doses of 16mg/kg and 32 mg/kg; from the results, the compound of the invention has better anti-infective activity in vivo and better therapeutic effect on COVID-19.

Claims

1. A diketone nitrogen heterocyclic compound with a structure shown in a general formula I or pharmaceutically acceptable salt and tautomer thereof, which is characterized in that the general formula I has the following structure:

wherein R ¹ is hydrogen, deuterium, C _1-6 alkyl;

R ² is halogen;

R ³ is halogen;

l is-NR ⁴ -or-NH-NH-;

R ⁴ is C _1-4 alkyl substituted by R ^4-1;

r ^4-1 is hydroxy;

The a end is connected with L, and the b end is connected with L Connected with the c terminal/>Are connected.

2. The diketone aza heterocyclic compound having a structure according to claim 1, wherein when R ¹ is C _1-6 alkyl, the C _1-6 alkyl is C _1-4 alkyl;

And/or, when R ⁴ is R ^4-1 substituted C _1-4 alkyl, said C _1-4 alkyl is methyl, ethyl or propyl.

3. The diketone aza ring compound having the structure shown in formula I or a pharmaceutically acceptable salt, tautomer thereof according to claim 1, wherein when R ¹ is C _1-6 alkyl, the C _1-6 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl;

and/or, when R ⁴ is C _1-4 alkyl substituted by R ^4-1, said C _1-4 alkyl substituted by R ^4-1 is

4. The diketone aza heterocyclic compound having a structure represented by formula I or a pharmaceutically acceptable salt, tautomer thereof according to claim 1, wherein R ¹ is hydrogen, deuterium, or C _1-6 alkyl;

R ² is halogen;

R ³ is halogen;

L is NR ⁴ -or-NH-NH-;

R ⁴ is C _1-4 alkyl substituted by R ^4-1;

r ^4-1 is hydroxy;

A is

5. The diketone nitrogen heterocyclic compound with a structure shown in a general formula I or pharmaceutically acceptable salts and tautomers thereof according to claim 1, wherein the compound shown in the general formula I is any one of the following compounds:

6. A process for the preparation of a diketone nitrogen heterocyclic compound having a structure as described in any one of claims 1-5, or a pharmaceutically acceptable salt, tautomer thereof, wherein compound II and compound III are reacted in a solvent under the action of a base/condensing agent, base/catalyst/ligand or base to form compound I;

Wherein X is amino, -NH-NH ₂, Y is halogen or C _1-3 alkylthio, Y is connected with the a end of A, R ¹、R²、R³、R⁴, L and A are as described in claims 1-5.

7. A pharmaceutical composition comprising a therapeutically effective amount of one or more diketone aza ring compounds having a structure according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, tautomer thereof, and a pharmaceutically acceptable carrier or adjuvant.

8. Use of a diketo-aza-cycle compound having a structure according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, tautomer thereof, for the preparation of a 3C-like cysteine protease inhibitor.

9. Use of a pharmaceutical composition according to claim 7 for the preparation of a 3C-like cysteine protease inhibitor.

10. Use of a diketo-aza ring compound having a structure according to any one of claims 1 to 5 or a pharmaceutically acceptable salt, tautomer thereof or a pharmaceutical composition according to claim 7 for the manufacture of a medicament for the treatment and/or prophylaxis of a viral infectious disease, wherein the virus comprises severe acute respiratory syndrome-related coronavirus-2 SARS-CoV-2, middle eastern respiratory syndrome-related coronavirus MERS-CoV, severe acute respiratory syndrome-related coronavirus SARS-CoV, influenza a virus, influenza b virus, enterovirus, coxsackie virus.